1. Field of the Invention
The present invention relates to a capillary electrophoretic apparatus for separating and analyzing a biopolymer such as protein or nucleic acid.
Such a capillary electrophoretic apparatus is applied to sequence determination for DNA. The capillary electrophoretic apparatus for DNA sequence determination electrophoreses a DNA fragment sample prepared by labeling a primer or a terminator with a fluorochrome and detects fluorescence from the DNA fragment sample during electrophoresis for determining the base sequence.
2. Description of the Prior Art
A DNA sequencer having high sensitivity, a high speed and high throughput is necessary for sequence determination for DNA such as a human genome having long base sequence. As an example, capillary electrophoresis employing a capillary column charged with a gel in place of slab gel electrophoresis employing a flat plate type slab gel is proposed. With such a capillary column, a sample can not only be readily handled or injected but also electrophoresed at a high speed and detected in high sensitivity as compared with the slab gel. If a high voltage is applied to the slab gel, a band is spread or a temperature gradient is caused due to influence by Joulean heat. However, the capillary column hardly causes such a problem and can perform detection in high sensitivity with small band spreading even if performing high-speed electrophoresis with application of a high voltage.
A multi-capillary electrophoretic apparatus prepared by arranging a plurality of capillary columns is also proposed.
An automatic DNA sequencer utilizes a fluorochrome for identifying the four types of bases forming DNA. A Rhodamine derivative such as R6G, R-110 or ROX or a fluorescein derivative such as FAM is utilized as the fluorochrome. An argon ion laser unit having dominant wavelengths of 488.0 nm and 514.5 nm is utilized as a laser beam source.
However, both of the wavelengths of 488.0 nm and 514.5 nm are separate from the absorption maximum wavelengths of the fluorescein derivatives and the Rhodamine derivatives. While fluorescein derivatives having an absorption maximum wavelength of 493.5 nm are not much reduced in efficiency, the Rhodamine derivatives, which are excited at 514.5 nm although its absorption maximum wavelength is 550 nm, has inferior efficiency.
In order to solve this problem, there has been made an attempt (referred to as an energy transfer method) of introducing both of the fluorescein derivative and the Rhodamine derivative into the same molecule when using the Rhodamine derivative as a label thereby improving the efficiency of the Rhodamine derivative through the principle of energy transfer.
The energy transfer method is superior to general methods, but yet has the following problems:
1. It is technically difficult to introduce a plurality of fluorochromes into the same molecule, and the cost is increased following this difficulty.
2. Extreme influence is exerted by Raman scattering since the excitation wavelength is in the visible region. When excited at 488 nm, a Raman scattering line of water around 516 nm forms background noise of a channel detecting the fluorescein derivative having a fluorescence maximum at 510 nm to reduce an S-N (signal-to-noise) ratio.
3. Influence is exerted by Rayleigh scattering to readily reduce the S-N ratio.
Considering a multi-capillary electrophoretic apparatus in which a plurality of capillary columns are so arranged that a plurality of samples are injected into the capillary columns and simultaneously electrophoresed in all capillary columns, one ends of the plurality of capillary columns defining a sample injection side are two-dimensionally arranged and fixed by a sample injection side holder while the other ends defining a detection side are aligned with each other on a plane and fixed by a detection side holder for forming a capillary array. The detection side holder is provided with a slot along the arrangement of the capillary columns, and parts of the capillary columns exposed through the slot define a part to be detected. When separating and detecting a sample containing a DNA fragment labeled in four types with a fluorescent material, excitation light is applied to the part to be detected for detecting fluorescence generated from sample components electrophoresed to the part to be detected thereby identifying the sample components.
The prior art employs an epi-optical system comprising a condenser lens condensing and projecting excitation light onto each capillary column on the part to be detected and receiving the fluorescence generated from the sample electrophoresed in the capillary column as an objective lens for projecting the excitation light and receiving the fluorescence through the same lens as an excitation-light receiving optical system. The objective lens is scanned along a straight line parallel to the plane of arrangement of the capillary columns on the part to be detected and perpendicular to the electrophoresis direction, thereby detecting the fluorescence as to all capillary columns.
In such an optical system, the objective lens is preferably arranged in proximity to the part to be detected for collecting the maximum amount of fluorescence in consideration of detection sensitivity. Therefore, the condenser lens having a short focal length is employed as the objective lens.
When employing the condenser lens having a short focal length as the objective lens, the amount of collected fluorescence is reduced to reduce the detection sensitivity if the position of the part to be detected of the capillary array slightly deviates in the direction of application of the excitation light. Therefore, high working accuracy is required when preparing the detection side holder and fixing the capillary columns to the detection side holder.
In the multi-capillary electrophoretic apparatus, the capillary columns are fixed to cassette holders on a sample introduction side and the detection side. The cassette holders two-dimensionally arrange the capillary columns on the sample introduction side and planarly align the same with each other on the detection side.
When charging each capillary column with a polymer, one end of the capillary column is stuck into and fixed to an elastic member such as a rubber stopper or fixed to a dedicated holder for polymer charging with an adhesive for filling up a clearance. The polymer is charged into the capillary column by fixing the elastic member or the dedicated holder to a vessel storing the polymer so that the end of the capillary column is dipped in the polymer, sealing the vessel and pressurizing the vessel with a pump for press-filling the polymer into the capillary column, or by connecting the elastic member or the dedicated holder to a pump, dipping another end of the capillary column into the polymer and decompressing the capillary column with the pump for inhaling the polymer into the capillary column.
When charging the polymer into the capillary column by press-filling or inhaling in the method of sticking and fixing the capillary column into and to the elastic member, pressure resistance of the elastic member may be so insufficient that the polymer cannot be smoothly charged into the capillary column. On the other hand, in the method of fixing the capillary column to the dedicated holder for polymer charging with an adhesive, it may be impossible to smoothly charge the polymer into the capillary column due to insufficient supply of the adhesive, to result in an inferior manufacturing yield.
A first objective of the present invention is to perform efficient detection in a capillary electrophoretic apparatus.
A second objective of the present invention is to provide a capillary cassette capable of reliably charging all capillary columns with polymers with a high yield in a multi-capillary electrophoretic apparatus.
A first aspect of the present invention for performing efficient detection comprises detection means exciting a fluorochrome bonded to a sample component as a label for making the same fluoresce and detecting the generated fluorescence without influence by Raman scattering or Rayleigh scattering. In a capillary electrophoretic apparatus according to the present invention, the detection means applies excitation light having a wavelength longer than the fluorescent wavelength of the fluorochrome, excites the fluorochrome by multiphoton absorption and detects fluorescence generated from the fluorochrome. In other words, this aspect utilizes a multiphoton absorption method of applying light (the excitation light having a longer wavelength than the fluorescent wavelength of the fluorochrome) having energy of one photon smaller than excitation energy for the fluorochrome to the sample bonded with the fluorochrome and making the fluorochrome absorb multiphotons thereby exciting the fluorochrome and making the same fluoresce.
In the multiphoton absorption method, both a fluorescein derivative and a Rhodamine derivative can be excited with a common laser wavelength. Therefore, it is not necessary to introduce a plurality of fluorochromes into the same molecule.
The excitation wavelength used in the multiphoton absorption method may be set in a range from 400 nm to 2 xcexcm, and preferably is set in the near infrared region of at least 600 nm. With the excitation wavelength of at least 600 nm, most of the Raman scattering line outgoing from the wavelength is Stokes Raman scattered light having a wavelength of at least 600 nm. Therefore, the Raman scattering light does not form background noise in detection of fluorescence from fluorescein or Rhodamine.
Furthermore, the intensity of Rayleigh scattering is in inverse proportion to the sixth power of the wavelength, and hence the excitation light of a longer wavelength region exceeding 600 nm utilized in the multiphoton absorption method is superior to an argon laser beam for suppressing Rayleigh scattering.
Thus, the fluorescence from the fluorochrome can be detected in a high S-N ratio while suppressing influence by Raman scattering and Rayleigh scattering by comprising the detection means employing the multiphoton absorption method, applying the light of a wavelength longer than the fluorescent wavelength of the fluorochrome to the fluorochrome for exciting the same and detecting the fluorescence thereof.
A second aspect of the present invention for performing efficient detection is to relieve requirement for working accuracy at the time of preparing a detection side holder and fixation of capillary columns to the detection holder, fix the position of a part to be detected of a capillary array to the apparatus with excellent reproducibility, and suppress reduction of detection sensitivity. in a multi-capillary electrophoretic apparatus The multi-capillary electrophoretic apparatus to which this aspect is applied comprises a capillary array in which a plurality of capillary columns are so arranged that one ends defining a sample injection side are fixed by a sample injection side holder, the other ends defining a detection side are aligned with each other on a plane and fixed by a detection side holder and a part to be detected is provided on the position of the detection side holder, a multi-capillary array electrophoresis part to which the sample injection side holder and the detection side holder are fixed so that samples are injected into the capillary columns, the ends on the sample injection side are dipped into a buffer solution, the ends on the detection side are dipped into another buffer solution and an electrophoresis voltage is applied through both buffer solutions for performing electrophoresis in all capillary columns, and a detection part applying light to the part to be detected of the capillary array and detecting light affected by interaction with the samples. According to this aspect, the multi-capillary array electrophoresis part includes a detection side holder fixing part fixing the detection side holder and a parallelism adjusting mechanism adjusting the parallelism between the detection part and the part to be detected.
Detection can be performed in constant sensitivity regardless of the position of the part to be detected by adjusting the parallelism between the detection part and the part to be detected by the parallelism adjusting mechanism.
The detection system of the detection part may be either a scanning system of sequentially detecting the capillary columns one by one on the part to be detected or an image system of collectively capturing the capillary columns on the part to be detected as an image.
The detection part in the scanning system comprises an epi-optical system condensing and projecting light onto one of the capillary columns on the part to be detected while receiving light affected by interaction with the samples and a scanning mechanism reciprocally moving the epi-optical system along a straight line parallel to the plane of arrangement on the part to be detected of the capillary array and perpendicular to the electrophoresis direction, and the parallelism adjusting mechanism adjusts the parallelism between a scanning axis of the epi-optical system and the part to be detected in this case.
In the parallelism adjustment, the scanning system fixes the detection side holder to the detection side holder fixing part, thereafter drives the scanning mechanism to reciprocate the epi-optical system in the direction perpendicular to the electrophoresis direction, and adjusts the parallelism between the scanning axis of the epi-optical system and the part to be detected by the parallelism adjusting mechanism on the basis of a current detection signal of the detection part.
The image system can be provided with an imaging optical system and a line sensor described in, for example, U.S. Pat. No. 5,534,703. In this case, the parallelism adjusting mechanism may adjust an optical axis of the imaging optical system.
A mode of the parallelism adjusting mechanism is preferably a gate adjusting mechanism adjusting a mounting angle of the detection side holder fixing part by rotation of a screw. Consequently, the parallelism between the scanning axis of the epi-optical system and the part to be detected can be adjusted in a simple structure through a simple operation.
Another mode of the parallelism adjusting mechanism preferably comprises an actuator automatically adjusting a gate angle of the detection side holder fixing part in correspondence to the detection signal at the time of scanning the epi-optical system. Consequently, a burden on an operator can be reduced.
Furthermore, the detection part preferably comprises an epi-optical system condensing and projecting light onto each capillary column on the part to be detected and receiving light affected by interaction with the samples, and a scanning mechanism reciprocally moving the epi-optical system along a straight line perpendicular to the electrophoresis direction while automatically adjusting the distance between the part to be detected and the epi-optical system in correspondence to a detection signal at the time scanning the same along the straight line. Consequently, the distance between the part to be detected and the epi-optical system can be rendered suitable without providing a parallelism adjusting mechanism.
The detection side holder fixing part preferably comprises a detection position member arranged between the part to be detected and the epi-optical system, having an opening on a position corresponding to the part to be detected and having a plane in contact with one surface of the part to be detected and a detected part pressing member having a plane pressing the part to be detected against the detection position member from a side opposite from the detection position member. Consequently, the plurality of capillary columns of the part to be detected can be fixed onto the plane of the detection position member with exceptional reproducibility.
The inventive multi-capillary electrophoretic apparatus according to this aspect fixes the part to be detected of the capillary array onto a plane of a movable plate by the detected part pressing member and thereafter adjusts a gate angle of the movable plate by a gate adjusting mechanism so that the parallelism between the part to be detected and the scanning axis of the epi-optical system can be adjusted, whereby requirement for working accuracy in preparation of the detection side holder and fixation of the capillary columns to the detection side holder can be relaxed, the position of the part to be detected can be fixed to the apparatus with excellent reproducibility, and reduction of detection sensitivity can be suppressed.
A capillary cassette according to the present invention capable of reliably charging all capillary columns with polymers in an excellent yield is a capillary cassette in which a plurality of capillary columns used in a multi-capillary electrophoretic apparatus are bundled so that first ends thereof are cylindrically bundled by a sleeve and clearances between the sleeve and the capillary columns and between the capillary columns are sealed with a filler.
The one ends of the capillary columns cylindrically bundled by the sleeve have a cylindrical outer shape and hence can be readily mounted on a polymer charger in an airtight manner, whereby the capillary columns can be readily charged with the polymers through a high pressure.
The sleeve is preferably prepared by shrinking a shrinkable member, and is preferably a heat-shrinkable tube.
The capillary column ends can be most densely and cylindrically bundled by passing the one ends of the plurality of capillary columns through the sleeve formed by a shrinkable member and thereafter shrinking the sleeve. When previously applying the filler to the capillary column surfaces on positions corresponding to the shrinkable member, the clearances between the capillary columns can be filled up with the filler without failure in the process of bundling the capillary columns. When employing a heat-shrinkable tube as the sleeve, the capillary column ends can be bundled by simply heating the same with a dryer or the like.
A mounting member for mounting the sleeve on the polymer charger in an airtight manner is preferably mounted on the sleeve.
The capillary column ends cylindrically bundled by the sleeve can be handled similarly to, for example, a pipe of a liquid chromatograph. For example, if a mounting member such as a ferrule, is mounted on the sleeve when charging the capillary columns with the polymers, the capillary column ends can be fixed to the polymer charger in an airtight manner.
It is preferable that a part to be detected in which the capillary columns are aligned with each other is provided on the side of the cylindrically bundled ends while the capillary columns are two-dimensionally arranged to define a sample injection part on the side of the other ends.
It is possible to dip the capillary column ends in various sample solutions respectively in sample injection for simultaneously injecting samples into the respective capillary columns by two-dimensionally arranging the capillary column ends opposite from the cylindrically bundled ends. Thus, the capillary columns can be reliably charged with the polymers in an exceptional yield. Furthermore, it is possible to apply a electrophoresis voltage across all capillary columns after sample injection for simultaneously separating and detecting the samples in the respective capillary columns.
The foregoing and other objects, features, aspects and advantages of the present invention will become more apparent from the following detailed description of the present invention when taken in conjunction with the accompanying drawings.